Red Star Active Dry Yeast (Link) was obtained. Several particles of yeast were aseptically spread onto LB Only agar and incubated at 30C. Isolated colonies were transferred to new LB Only agar and continued to incubate. A total of 20 mL of 10 mM Tris was added to the surface of an LB Only plate containing yeast growth (plate seen below). Tris was added in 10 mL increments, plates were scraped using L-shaped cell spreaders, and solutions were pipetted off the surface and transferred to a 50 mL conical tube.
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| Yeast on LB |
A small portion of the Yeast Stock was diluted 1:100 in ddH2O and 10 uL were transferred to a hemocytometer for quantification. Cells were counted and averages were determined (48 cells per small square).
Hemocytometer Equation:
Yeast stock was determined to be at 4.8x10^7 cells/mL.
There will be six different treatment groups for the upcoming experiment:
A. BAD only
B. BAD + 1000 cells/mL Yeast
C. BAD + Antioxidant
D. BAD + 1000 CFU/mL P. larvae
E. BAD + 1000 CFU/mL P. larvae + Antioxidant
F. BAD + 1000 CFU/mL P. larvae + 1000 cells/mL Yeast
BAD + Antioxidants (Treatment C) was provided to me from the USDA as diluted by Garret S. for Anna B.'s antioxidant/wax project.
Dilutions:
Treatment B: Yeast Stock was diluted 1:10 down to 4.8x10^4 cells/mL in ddH2O (100 uL + 900 uL H2O). 11 uL of this 10^4 dilution was added to 489 uL of BAD to create 1056 CFU/mL Yeast spiked BAD treatment group
Treatment D: P. larvae Spore Stock A (3.05x10^6 CFU/mL) was diluted 1:10 down to 3.05x10^4 CFU/mL in ddH2O (100 uL + 900 uL H2O). 16 uL of this 10^4 dilution was added to 484 uL of BAD to create 976 CFU/mL P. larvae spiked BAD.
Treatment E: Same procedure as Treatment D, except BAD containing the antioxidant was used.
Treatment F: 11 uL of the 10^4 Yeast dilution (Treatment B) and 16 uL of the 10^4 P. larvae dilution (Treatment D) were added to 473 uL of BAD.
Dilutions were stored at 4C overnight. They will be applied to 1st instar honey bee larvae in about 16 hours after creation.
//EWW
Hemocytometer Equation:
Cells Counted * (dilution factor / number of small squares counted) * 10,000 = Cells / mL
or
Average cells per small square * dilution factor * 10,000 = Cells / mL
Yeast stock was determined to be at 4.8x10^7 cells/mL.
There will be six different treatment groups for the upcoming experiment:
A. BAD only
B. BAD + 1000 cells/mL Yeast
C. BAD + Antioxidant
D. BAD + 1000 CFU/mL P. larvae
E. BAD + 1000 CFU/mL P. larvae + Antioxidant
F. BAD + 1000 CFU/mL P. larvae + 1000 cells/mL Yeast
BAD + Antioxidants (Treatment C) was provided to me from the USDA as diluted by Garret S. for Anna B.'s antioxidant/wax project.
Dilutions:
Treatment B: Yeast Stock was diluted 1:10 down to 4.8x10^4 cells/mL in ddH2O (100 uL + 900 uL H2O). 11 uL of this 10^4 dilution was added to 489 uL of BAD to create 1056 CFU/mL Yeast spiked BAD treatment group
Treatment D: P. larvae Spore Stock A (3.05x10^6 CFU/mL) was diluted 1:10 down to 3.05x10^4 CFU/mL in ddH2O (100 uL + 900 uL H2O). 16 uL of this 10^4 dilution was added to 484 uL of BAD to create 976 CFU/mL P. larvae spiked BAD.
Treatment E: Same procedure as Treatment D, except BAD containing the antioxidant was used.
Treatment F: 11 uL of the 10^4 Yeast dilution (Treatment B) and 16 uL of the 10^4 P. larvae dilution (Treatment D) were added to 473 uL of BAD.
Dilutions were stored at 4C overnight. They will be applied to 1st instar honey bee larvae in about 16 hours after creation.
//EWW
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